This project concerns an investigation of whether or not insulin and/or insulin-like polypeptides present in brain and other extrapancreatic tissues can be de novo synthesized in situ. We have used as a probe a cloned cDNA fragment of proinsulin to assess the presence of mRNA in various tissues that can hybridize to the DNA probe. mRNA from various tissues of rat (brain and 14 peripheral tissues) and from the brain of pig, cow and human was extracted, fractionated by agarose gel electrophoresis, blotted to nitrocellulose membranes and hybridized with the radioactivity-labeled cDNA probe. We found that there are at least five mRNA species hybridizing to prbe (with sizes of 560, 630, 860, 1200 and 1500 nucleotides) in the extrapancreatic tissues, while in the pancreas, only the two species with the smallest size can be found. The specific hybridization is strong in the olfactory bulb and tubercle, hypothalamus, hippocampus, brain stem, frontal cortex and cerebellum but is weak in the posterior cortex and spinal cord. Moreover, we found that mRNA from human brain shows two hybidization bands with sizes of 860 and 2000 nucleotides and mRNA from brains of pig and cow shows three hybridization bands with sizes of 1370, 2660 and 3770 nucleotides. These results may suggest that transcription of genes for insulin and/or insulin-like polypeptides occur in the brain and other extrapancreatic tissues; however, the transcriptional products are highly heterogenous. Further studies are in progress to verify the biosynthesis of these polypepties in extrapancreatic tissues.